Diabetes has become an increasingly important health issue. A serious complication of diabetes is impaired oral wound healing, which negatively affects the response to trauma and surgery. Despite being identified by NIH as an important health issue there have been surprisingly few studies that have examined mechanisms by which oral connective tissue healing is impaired by diabetes. This is significant since oral wound healing is subject to unique stresses associated with the oral cavity including bacteria which bathe the mucosal surfaces and forces of mastication, amongst others. Preliminary data demonstrate that oral wounds have impaired healing and that they exhibit increased FOXO1 DNA binding activity and nuclear translocation in fibroblastic cells. Based upon Preliminary Data and published reports increased FOXO1 in diabetic wounds could interfere with the wound healing process by increasing fibroblast apoptosis, decreasing fibroblast proliferation and enhancing the expression of pro- inflammatory mediators. The potential role of FOXO1 in oral diabetic wound healing will be studied in Aim 1 in transgenic mice with floxed FOXO1 that will be deleted in adult diabetic and normoglycemic mice by tamoxifen induced Cre recombinase. Moreover, Cre recombinase will be restricted to cells of the fibroblast lineage, which will avoid the pitfalls of global FOXO1 deletion. By this approach we will determine whether diabetes-enhanced FOXO1 upregulation in fibroblasts contributes to altered diabetic healing. By investigating genes that are modulated by FOXO1 deletion we will also examine specific downstream targets through which FOXO1 may affect the healing process. Aim 2 will investigate three specific mechanisms for diabetes enhanced FOXO1. These include increased TNF-a, greater signaling of advanced glycation end products through RAGE and the impact of oral bacteria. This will be accomplished by the use of specific inhibitors of TNF and of RAGE and antibiotics to decrease the bacterial burden. These mechanisms are consistent with Preliminary Data which indicate that TNF and RAGE signaling stimulate FOXO1 activation in fibroblasts in vitro and that oral wound healing can be improved in immunodeficient animals through antibiotic treatment. In summary, we propose to test the hypothesis that diabetes enhanced FOXO1 interferes with oral wound healing and to investigate mechanisms through which diabetes leads to greater FOXO1 in fibroblastic cells. These studies will investigate a novel mechanism for diabetes impaired oral wound healing and one that has not been investigated previously in any form of wound healing. We expect that they will provide a better understanding of the steps that lead to this diabetic complication. PUBLIC HEALTH RELEVANCE: The goal of the proposed studies is to investigate mechanisms by which diabetes interferes with the oral wound healing through increased FOXO1. This is based on Preliminary Data and published reports that FOXO1 stimulates fibroblast apoptosis, reduces proliferation and increases the expression of inflammatory mediators. This is striking since diabetic wound healing is characterized by greater fibroblast apoptosis and reduced proliferation as well as prolonged inflammation. Three potential mechanisms for diabetes enhanced FOXO1 will be examined including greater levels of TNF-a, signaling of advanced glycation end products through RAGE and the effect of oral bacteria.